WO2014205555A1 - Procédés et utilisations pour diagnostiquer et traiter le cancer de la prostate - Google Patents

Procédés et utilisations pour diagnostiquer et traiter le cancer de la prostate Download PDF

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WO2014205555A1
WO2014205555A1 PCT/CA2014/000538 CA2014000538W WO2014205555A1 WO 2014205555 A1 WO2014205555 A1 WO 2014205555A1 CA 2014000538 W CA2014000538 W CA 2014000538W WO 2014205555 A1 WO2014205555 A1 WO 2014205555A1
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pcat18
prostate cancer
expression level
pca
subject
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WO2014205555A8 (fr
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Cheryl D. HELGASON
Francesco Crea
Yuzhuo WANG
Kim N. CHI
Akira WATAHIKI
Hui Hsuan LIU
Abhijit PAROLIA
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British Columbia Cancer Agency Branch
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Priority to US14/901,618 priority Critical patent/US20180051340A1/en
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Publication of WO2014205555A8 publication Critical patent/WO2014205555A8/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a novel biomarker for prostate cancer.
  • the present invention relates to methods and uses of a novel long non-coding RNA (IncRNA), termed PCAT18, for the early detection, diagnosis, prognosis, classification, treatment monitoring, or treatment of prostate cancer (PCa).
  • IncRNA novel long non-coding RNA
  • PCa prostate cancer
  • mCRPC metastatic castration-resistant PCa
  • RNA molecules produced in human cells are not translated, and thus protein-coding genes account for only a small percentage of all RNAs (3).
  • These non-coding transcripts include the well- known ribosomal-, transfer- and micro-RNAs (rRNA, tRNA, miRNA respectively).
  • MiRNA profiling in tumor specimens and patient-derived biological fluids is emerging as a powerful tool to differentiate localized and metastatic PCa (4).
  • a less investigated class of non-coding RNAs is represented by long non-coding RNAs (IncRNAs), i.e. transcripts longer than 200bp with no protein-coding function (5). Recent evidence indicates that IncRNAs may be an overlooked source of cancer biomarkers and therapeutic targets.
  • IncRNA has been used as a catch-all definition, including poly-adenylated and non-poly-adenylated sequences, as well as intergenic and intronic transcripts. Estimates suggest the number of human IncRNAs rivals the count of protein-coding genes, ranging from 10,000 to 20,000 (6). Despite these large numbers, only a handful of IncRNAs have been characterized. Notably, most characterized IncRNAs display deregulated expression in cancer cells, where they play oncogenic or tumor suppressive functions (6). A striking feature of some IncRNAs is their tissue-specificity which prompted some authors to propose them as novel biomarkers (6).
  • PCGEM1 and PCA3 Two previously characterized IncRNAs (PCGEM1 and PCA3) are specifically expressed in PCa compared to an array of normal and neoplastic tissues (7, 8).
  • PCA3 is present in urine samples from PCa patients and is able to detect the disease with 77.5% sensitivity and 57.1% specificity (9).
  • PCA3 levels are not able to discriminate between indolent and clinically aggressive PCa (9).
  • the clinical utility of PCGEM1 has also not been determined. Accordingly, it is unclear whether PCA3 or PCGEM1 is a viable therapeutic target. [0004]
  • a new diagnostic, prognostic and therapeutic biomarker is, therefore, needed for early recognition, detection, diagnosis and effective management of PCa.
  • such a biomarker should be able to distinguish between localized, indolent PCa and clinically aggressive PCa and detectable in a subject's blood, urine, saliva, plasma or tissue. It would be especially useful to have a biomarker that can identify those subjects whose prostate cancers are at an elevated risk for progression or transformation to life-threatening androgen- resistant or metastatic disease.
  • the present invention relates generally to methods and uses of diagnosing, determining risk of developing, prognosing, monitoring treatment of, detecting, classifying and treating prostate cancer in a subject suspected of having or having prostate cancer by assessing the expression level of PCAT18.
  • PCAT18 RNA is a long noncoding RNA identified herein as being differentially expressed in cancer calls, particularly in prostate cancer cells, as compared to normal prostate cells and as being specific for prostate cancer as compared to other neoplasms.
  • the present invention relates to method for diagnosing prostate cancer in a subject suspected of having prostate cancer comprising: (a) assessing the expression level of PCAT18 in a biological sample obtained from the subject; (b) comparing the expression level of PCAT18 in the biological sample to a reference expression level; and (c) identifying the subject as having prostate cancer when the expression level of PCAT18 in the biological sample is greater than the reference expression level, or identifying the subject as not having prostate cancer when the expression level of PCAT18 in the biological sample is not greater than the reference expression level.
  • the present invention relates to a method for determining the risk of a subject for developing prostate cancer comprising: (a) assessing the expression level of PCAT18 in a biological sample obtained from the subject; (b) comparing the expression level of PCAT18 in the biological sample to a reference expression level; and (c) identifying the subject as having an increased risk of developing prostate cancer when the expression level of PCAT18 in the biological sample is greater than the reference expression level, or identifying the subject as not having an increased risk of developing prostate cancer when the expression level of PCAT18 in the biological sample is not greater than the reference expression level.
  • the present invention relates to a method for monitoring a treatment for prostate cancer in a subject diagnosed with prostate cancer comprising: (a) obtaining a baseline level by assessing the expression level of PCAT18 in a biological sample obtained from the subject prior to administration of the treatment; (b) administering the treatment to the subject for a treatment period; (c) after the treatment period, assessing the expression level of PCAT18 in a second biological sample obtained from the subject; (d) comparing the expression level of PCAT18 in the second biological sample to the baseline level; and (e) identifying a poor response to the treatment when the expression level of PCAT18 in the second biological sample is greater than the baseline level, or identifying a good response to the treatment when the expression level of PCAT18 in the second biological sample is not greater than the baseline level.
  • the present invention further relates to a method for determining a prognosis of a subject diagnosed with having prostate cancer comprising: (a) assessing the expression level of PCAT18 in a biological sample obtained from the subject; (b) comparing the expression level of PCAT18 in the biological sample to a threshold expression level; and (c) determining a prognosis for the subject diagnosed with having prostate cancer based on the expression level of PCAT18 in the biological sample relative to the threshold expression level.
  • the present invention relates to a method for determining a risk of metastatic spread of prostate cancer in a subject diagnosed with prostate cancer comprising: (a) assessing the expression level of PCAT18 in a biological sample obtained from the subject; (b) comparing the expression level of PCAT18 in the biological sample to a threshold expression level; and (c) identifying the subject as having an increased risk of metastatic spread when the expression level of PCAT18 in the biological sample is greater than the threshold expression level, or identifying the subject as not having an increased risk of metastatic spread when the expression level of PCAT18 in the biological sample is not greater than the threshold expression level.
  • the biological sample may be plasma, blood, serum, urine, saliva or tissue obtained from the subject.
  • the tissue may comprise a cancerous prostate tissue sample, a benign prostatic hyperplasia tissue, or a normal prostate tissue.
  • the assessing of the expression level of PCAT18 in the biological samples obtained from subjects may be performed by evaluating the amount of PCAT18 RNA in the biological samples.
  • the present invention relates to a method of treating a subject diagnosed with prostate cancer by administering a therapeutically effective amount of an inhibiting agent of PCAT18, wherein the inhibiting agent of PCAT18 is an antisense oligonucleotide, an siRNA, or a combination thereof.
  • the siRNA used in the method of treating described above may comprise an antisense nucleotide sequence corresponding to SEQ ID NO:22 or SEQ ID NO:23.
  • the antisense oligonucleotide used in the method of treating described above comprises a nucleotide sequence corresponding to SEQ ID NO:24 or SEQ ID NO:25.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutic agent effective to reduce an amount of PCAT18 in cancerous prostate cells exposed to the therapeutic agent, and a pharmaceutically acceptable carrier, wherein the therapeutic agent is an antisense oligonucleotide, an siRNA, or a combination thereof.
  • the siRNA used in the pharmaceutical composition described above may comprise an antisense nucleotide sequence corresponding to SEQ ID NO:22 or SEQ ID NO:23.
  • the antisense oligonucleotide used in the pharmaceutical composition described above may comprise a nucleotide sequence corresponding to SEQ ID NO:24 or SEQ ID NO:25.
  • the present invention relates to a use of PCAT18 RNA for diagnosing prostate cancer in a subject suspected of having prostate cancer, wherein PCAT18 RNA comprises a nucleotide sequence corresponding to SEQ ID ⁇ . ⁇ .
  • the present invention relates to a use of PCAT18 RNA for determining the risk of a subject in developing prostate cancer, wherein PCAT18 RNA comprises a nucleotide sequence corresponding to SEQ ID NO:1.
  • the present invention relates to a use of PCAT18 RNA for monitoring a treatment for prostate cancer in a subject diagnosed with prostate cancer, wherein PCAT18 RNA comprises a nucleotide sequence corresponding to SEQ ID NO:1.
  • the present invention further relates to a use of an inhibiting agent of
  • PCAT18 RNA for treating a subject diagnosed with prostate cancer, wherein PCAT18 RNA comprises a nucleotide sequence corresponding to SEQ ID NO: 1.
  • IncRNA LOC728606
  • PCAT18 a novel use of a IncRNA (LOC728606), herein termed JUPITER or PCAT18, as a biomarker for diagnosis and prognosis of prostate cancer is provided.
  • JUPITER or PCAT18 as a target for development of therapies for treatment of prostate cancer (including but not limited to localized, invasive, androgen (castration) resistant and metastatic prostate cancer) is provided.
  • IncRNA LOC728606
  • PCAT18 a novel use of a IncRNA (LOC728606), herein termed JUPITER or PCAT18, as a biomarker for the early detection of prostate cancer is provided.
  • IncRNA LOC728606
  • PCAT18 a novel use of a IncRNA (LOC728606), herein termed JUPITER or PCAT18, as a biomarker for the diagnosis of prostate cancer.
  • JUPITER herein termed JUPITER or PCAT18, as a biomarker for the prognosis of prostate cancer, whereby increased levels of JUPITER measured in samples obtained from a patient with prostate cancer is predictive of poorer disease outcome or increased risk of disease relapse is provided.
  • a novel use of a IncRNA herein termed JUPITER or PCAT18, is provided as a biomarker for assessment of the metastatic potential of a prostate tumour whereby measurement of increased levels (relative to sampling at earlier timepoints) of JUPITER in samples from a subject with prostate cancer is indicative of increased risk or potential for metastatic spread (metastasis).
  • a novel use of a IncRNA herein termed JUPITER or PCAT 18, is provided as a biomarker for detection of prostate cancer at increased risk of progression to or that has already progressed to the stage of androgen (castration) resistant disease.
  • JUPITER herein termed JUPITER or PCAT18
  • JUPITER is provided as a biomarker that may be used in combination with other prostate cancer biomarkers (including but not limited to PSA, PCGEM1 , PCA3 etc.) in tests or methods for the detection, prognosis or treatment monitoring of prostate cancer.
  • a novel use of a IncRNA including but not limited to PSA, PCGEM1 , PCA3 etc.
  • IncRNA as a prostate cancer biomarker useful in tests/assays for monitoring the outcome of patients with prostate cancer (treatment response) that are treated with curative intent.
  • IncRNA herein termed JUPITER or PCAT18
  • a novel use of a IncRNA as a prostate cancer biomarker i.e.
  • the nucleotide sequence of the IncRNA is of about 90 % or greater similarity to the sequence of JUPITER (LOC728606) (SEQ ID NO:1 ).
  • a novel use of a IncRNA as a prostate cancer biomarker i.e.
  • the nucleotide sequence of the IncRNA is of about 95 % or greater similarity to the sequence of JUPITER (LOC728606) (SEQ ID NO: 1 ).
  • a novel use of a IncRNA as a prostate cancer biomarker i.e.
  • IncRNA as a prostate cancer biomarker (i.e. including but not limited to use of said biomarker for detection, diagnostic, prognostic or treatment-monitoring) or target for treatment of prostate cancer is provided, whereby the IncRNA comprises a contiguous nucleotide sequence of at least 200 base-pairs in length and whereby said IncRNA comprises a 200 base-pair (or longer) nucleotide sequence that is a fragment of the nucleotide sequence of JUPITER (LOC728606) (SEQ ID NO: 1 ).
  • JUPITER LOC728606
  • Figure 1 Hematoxylin-eosin staining of the prostate cancer xenograft
  • LTL-313H cells are more locally invasive to the adjacent kidney than LTL-313B cells, and show signs of distant metastatic spreading (never found in LTL-313B-engrafted mice).
  • B Quantitative PCT (qPCR) confirmation of RNA sequencing data (columns represent average value, bars represent standard deviation, 2 replicate experiments). Values indicate relative expression level in LTL-313H vs. LTL-313B cells (i.e., the fold change of 313H/313B).
  • the 4 most up- regulated transcripts were chosen (LOC728606, PCGEM1 , H19, LINC461_1 ), along with 3 randomly selected transcripts (LOC285419, NCRNA1 16, LINC461_3).
  • C Schematic representation of the PCAT18 locus (NLM "Gene” website). The gene is located in a region between 24,286 and 24,266 K (Chromosome 18 primary assembly). Lines represent introns, rectangles represent exons. Dotted lines represent a relative distance that is larger than the one shown in the schematic representation. Arrows represent transcription direction.
  • ORF open reading frame finder output for PCAT18 sequence. Open Reading Frames are shown as shaded squares throughout the sequence. Each lane represents a possible reading frame. The software identified no ORF longer than 267 bp for a transcript longer than 2Kb. Considering 6 possible reading frames, protein-coding regions could account for no more than 16% of the whole transcript.
  • C PCAT18 expression in various prostate cancer cell lines (22RV1 ; LNCaP; human prostate cancer cell line, C4-2; PC3; and H660) relative to that in a benign prostatic hyperplasia cell line (BPH1).
  • D siRNA-mediated PCAT18 silencing using two PCAT18-specific siRNAs (siRNA 1 and siRNA2) compared to a control.
  • E Cell growth inhibition in the human prostate cancer cell line (C4-2) after specific silencing of PCAT18 expression using two PCAT18-specific siRNAs (siRNA 1 and siRNA2). Compared to a negative control (NC).
  • LOC728606 (PCAT18) down-regulation is comparable to PSA.
  • Data are from LTL-331 xenografts human prostate cancer xenografts (www.livingtumorlab.com) and normalized to the average HPRT1 expression level in testosterone-supplemented animals.
  • HPRT1 expression is stable pre- and post-castration (unpublished microarray data).
  • Figure 5 shows the nucleotide sequence of the PCAT18 transcript (SEQ ID NO:1) (Entrez Gene ID: 728606; RefSeq ID: NR_024259.1 ).
  • FIG. 6 PCAT18 expression levels in untreated LNCaP cells (Control) and cells supplemented with dihydrotestosterone (DHT, 10nM, 6-24-48h). LNCaP cells were grown in phenol red-free medium (RPMI-1640) supplemented with 10% charcoal-stripped FBS. Columns represent mean value (2 independent experiments performed in triplicate), bars standard deviation.
  • B The living tumor lab (www.livingtumorlab.com) comprises a collection of patient-derived PCa tumor tissue xenografts, originated with a method described in ref.
  • LTL313B An androgen-dependent PCa line (LTL313B) has been exposed to castrate-levels of testosterone for a prolonged time, in order to generate a castration-resistant subline.
  • the figures show LTL313B tumor volume (B) and serum PSA levels (C) before and after castration.
  • Neoplastic cells were implanted in male NOD/SCID intact mice, supplemented with testosterone until castration. Serum PSA was measured and mice were sacrificed for tumor volume measurement at indicated time points, as described before (Lin D, et al. High fidelity patient-derived xenografts for accelerating prostate cancer discovery and drug development. Cancer research. 2013).
  • a castration-resistant, AR-positive cell line was generated (LTL-313BR).
  • FIG. 7 (A) C4-2 invasion was quantified 24h after the start of the invasion assay. Cells were transfected with 2nM Negative Control (NC) or PCAT18-targeting siRNAI and siRNA2. Columns represent mean value (4 experiments) bar SD. *** p ⁇ 0.001 (ANOVA and Dunnett's post-test). (B) C4-2 cell migration was quantified at 6h, 24h or 48h post- transfection, ** P ⁇ 0.01 , * ** P ⁇ 0.001 (siRNA vs.
  • NC 2 way ANOVA and Tukey's post-test.
  • C LNCaP
  • C4-2 D
  • BPH BPH
  • PCAT18-targeting siRNAs both at 2nM concentration
  • LNCaP cells were transfected with negative control (NC) or PCAT18-targeting siRNAs for 5 days. Bars represent mean values, lines standard deviations (2 independent experiments performed in triplicate). *** p ⁇ 0.001 with respect to NC (ANOVA and Dunnet's post-test).
  • Figure 8 (A) TaqMan qPCR confirmation of PCAT18 expression in PCa xenograft models. (B) TaqMan qPCR confirmation of PCAT 8 expression in clinical samples. (C) Basal expression levels of PCAT18 in a panel of prostate cancer cell lines. Columns represent mean values, bars standard deviations (2 independent experiments). (D) Subcellular localization of PCAT18, GAPDH and MALAT1. Cellular (C) and Nuclear (N) RNA fractions where extracted and quantified by TaqMan assay, as described in methods section of the Examples. Columns represent mean value, bars standard deviation (2 independent experiments). (E) TaqMan qPCR confirmation of siRNA-mediated PCAT18 silencing (C4-2 cells). Columns represent mean value, bars standard deviation (2 independent experiments). [0047] Figure 9 shows the nucleotide sequences of the antisense oligonucleotides.
  • A shows the nucleotide sequence of antisense oligonucleotide (NC) with no known specific target in human or mouse genome (SEQ ID NO:26).
  • B shows the nucleotide sequence of antisense oligonucleotide AS02 (SEQ ID NO:24).
  • C shows the nucleotide sequence of antisense oligonucleotide AS07 (SEQ ID NO:25).
  • Figure 10 shows the results of PCAT18 knockdown in C4-2 cells using antisense oligonucleotides AS02 and AS07 corresponding to SEQ ID NO:24 and SEQ ID NO:25, respectively, and using antisense oligonucleotide (NC) with no known specific target in human or mouse genome (corresponding to SEQ ID NO: 26).
  • NC antisense oligonucleotide
  • the columns and the bars represent mean value and standard deviation, respectively. **** p ⁇ 0.0001 (ANOVA and Dunnetts's post-test).
  • the present invention relates to a long noncoding RNA (IncRNA) and methods and uses of the IncRNA for diagnosing, prognosing, monitoring and treating PCa.
  • IncRNA long noncoding RNA
  • IncRNAs may be transcribed from any genomic region, including, but not limited to, intergenic IncRNA or intervening non-coding RNA (lincRNA), which refers to IncRNA transcripts that are located between two protein-coding genes and transcribed from the + and/or -DNA strand(s); and intragenic IncRNA, which refers to IncRNA transcripts that are located within a protein-coding gene.
  • Intragenic IncRNAs may be located within a coding region (i.e., an exon) of the gene and/or within a non-coding region (i.e., an intron) of the protein-coding gene, and transcribed from the + and/ or -DNA strand(s).
  • the present invention relates generally to identifying and characterizing long noncoding RNAs ("IncRNAs”) that are differentially expressed in cancer cells, particularly in prostate cancer cells, as compared to normal prostate cells.
  • IncRNAs long noncoding RNAs
  • PCAT18 Prostate Cancer-Associated Transcript-18; also referred to herein as JUPITER
  • JUPITER Prostate Cancer-Associated Transcript-18
  • PCAT18 whose expression is: (1 ) significantly higher in PCa compared to 26 other benign and neoplastic tissues; (2) detectable in plasma samples; (3) able to discriminate between localized disease and mCRPC, as described further below.
  • the term “about” refers to an approximately +/-10% variation from a given value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.
  • the term “plurality” as used herein means more than one, for example, two or more, three or more, four or more, and the like.
  • compositions may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one” and “one or more than one.”
  • the terms “comprising,” “having,” “including” and “containing,” and grammatical variations thereof, are inclusive or open-ended and do not exclude additional, un-recited elements and/or method steps.
  • the term “consisting essentially of when used herein in connection with a composition, use or method, denotes that additional elements and/or method steps may be present, but that these additions do not materially affect the manner in which the recited composition, method or use functions.
  • compositions, use or method excludes the presence of additional elements and/or method steps.
  • a composition, use or method described herein as comprising certain elements and/or steps may also, in certain embodiments consist essentially of those elements and/or steps, and in other embodiments consist of those elements and/or steps, whether or not these embodiments are specifically referred to.
  • localized prostate cancer or "primary prostate cancer” refers to prostate cancer that is only in the prostate gland and has not metastasized or spread to another part of the body.
  • An expression level of PCAT18 in a biological sample that is between about a 1.1 fold-change and about a 4 fold-change over the reference expression level, or any amount therebetween is indicative of primary prostate cancer.
  • metastatic prostate cancer refers to prostate cancer that has metastasized or spread outside the prostate gland to the lymph nodes, bones or other areas of the body.
  • An expression level of PCAT18 in a biological sample that is greater than about a 4 fold-change over the reference expression level, for example greater than about 4 fold to about 1000 fold, or any amount therebetween is indicative of metastatic prostate cancer, or metastatic castration-resistant prostate cancer.
  • mCPRC metal-static castration-resistant prostate cancer
  • the progression of PCa may be classified using several methods including measuring PSA levels, Gleason Score, tumour stage typing, or a combination thereof (see for example, www.cancer.gov/cancertopics/treatment/prostate/understanding-prostate-cancer- treatment/page3).
  • low risk PCa may be defined as having a Gleason Score of 6 or lower (tumour stage T1 or T2a)
  • a medium-risk PCa may be defined as having a Gleason Score of 7 (tumour stage T2b)
  • a high risk PCa may be defined as having a Gleason Score of 8 or higher (tumour stage T2c; Mazhar & Waxman.
  • a low Gleason PCa as used herein is characterized as having a Gleason Score of less than 6.
  • a more aggressive PCa; as used herein is characterized as having a Gleason Score of 7 or greater than 7 (i.e. medium risk and high risk prostate cancer).
  • an "expression level" of a transcript in a subject refers to an amount of transcript, such as PCAT18 RNA, in the subject's undiagnosed biological sample.
  • the expression level may be compared to a reference expression level to determine a status of the sample.
  • a subject's expression level can be either in absolute amount (e.g., number of copies/ml, nanogram/ml or microgram/ml) or a relative amount (e.g., relative intensity of signals; a percent or "fold” or "fold-change” increase).
  • a “reference level” or “reference expression level” (may also be considered a control), as used herein refers to an amount of the PCAT18 RNA or a range of amounts of the PCAT18 RNA measured in a normal individual or in a population of individuals without prostate cancer.
  • a reference expression level of the PCAT18 may be determined based on the expression level of PCAT18 in samples obtained from normal individuals.
  • a reference expression level can be either in absolute amount (e.g., number of copies/ml, nanogram/ml or microgram/ml) or a relative amount (e.g., relative intensity of signals; a percent or "fold” or “fold-change” increase).
  • a “threshold level” or “threshold expression level” refers to an expression level of PCAT18 in a biological sample that is between about a 1.1 fold-change and about a 4 fold-change over the reference expression level, or any amount therebetween.
  • a threshold expression level is indicative of localized prostate cancer or primary prostate cancer.
  • a “baseline level” refers to an expression level of PCAT18 in a first biological sample obtained from a subject that is determined prior to any treatment or during any treatment, and is used as comparison to a second expression level of PCAT18 that is assessed from a second biological sample that is obtained from the subject at a time after the first biological sample is obtained.
  • This baseline level may be used, for example, without limitation, in monitoring the progression of PCa in a subject, monitoring a treatment regimen or treatment modality in a subject having PCa, determining whether a treatment regimen or treatment modality should be considered in a subject, determining whether a treatment regimen or treatment modality should be discontinued in a subject, or determining whether a treatment regimen or treatment modality should be modified in a subject.
  • normal individual refers to an individual that has been tested for prostate cancer using a combination of diagnostic methods, including T stage, Gleason grade, plasma PSA levels and PCAT18 expression levels and determined to not have prostate cancer by a physician.
  • gene refers to a segment of nucleic acid that encodes an RNA, which RNA can be a coding or noncoding RNA.
  • the term "selectively hybridize,” as used herein, refers to the ability of a particular nucleic acid sequence to bind detectably and specifically to a second nucleic acid sequence. Selective hybridization generally takes place under hybridization and wash conditions that minimize appreciable amounts of detectable binding to non-specific nucleic acids. High stringency conditions can be used to achieve selective hybridization conditions as known in the art and discussed herein. Typically, hybridization and washing conditions are performed at high stringency according to conventional hybridization procedures with washing conditions utilising a solution comprising 1-3 x SSC, 0.1-1 % SDS at 50-70°C, with a change of wash solution after about 5-30 minutes.
  • identity refers to the measure of the identity of sequence between two nucleic acids molecules. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. Two nucleic acid sequences are considered substantially identical if they share at least about 80% sequence identity or at least about 81% sequence identity, or at least about 82% sequence identity, or at least about 83% sequence identity, or at least about 84% sequence identity, or at least about 85% sequence identity, or at least about 86% sequence identity, or at least about 87% sequence identity, or at least about 88% sequence identity, or at least about 89% sequence identity, or at least about 90% sequence identity.
  • two nucleic acid sequences are considered substantially identical if they share at least about 91% sequence identity, or at least about 92% sequence identity, or at least about 93% sequence identity, or at least about 94% sequence identity, or at least about 95% sequence identity, or at least about 96% sequence identity, or at least about 97% sequence identity, or at least about 98% sequence identity, or at least about 99% sequence identity.
  • Sequence identity may be determined by the BLAST algorithm which was originally described in Altschul et al. (1990) J. Mol. Biol. 215:403-410.
  • the BLAST algorithm may be used with the published default settings.
  • the degree of identity between sequences is a function of the number of matching positions shared by the sequences and the degree of overlap between the sequences.
  • SEQ ID NO: 1 or a contiguous portion of SEQ ID NO:1 it is intended that the equivalent number of nucleotides be compared to SEQ ID ⁇ . or the contiguous portion of SEQ ID NO: 1 , respectively.
  • sequence identity of a given sequence may be calculated over the length of the reference sequence (i.e., SEQ ID NO:1 or the contiguous portion of SEQ ID NO: 1 ).
  • corresponding to or “corresponds to” indicate that a polynucleotide sequence is identical to all or a portion of a reference polynucleotide sequence.
  • the term “complementary to” is used herein to indicate that the polynucleotide sequence is identical to all or a portion of the complementary strand of a reference polynucleotide sequence.
  • the nucleotide sequence "TATAC” corresponds to a reference sequence "TATAC” and is complementary to a reference sequence "GTATA.”
  • target gene refers to the gene the expression of which is to be modulated with a siRNA molecule or ASO molecule or other inhibiting agent of the present invention.
  • the target gene is the IncRNA locus LOC728606 or the PCAT18 gene.
  • target RNA refers to the RNA transcribed from a target gene.
  • antisense strand refers to a nucleotide sequence that is complementary to the nucleotide sequence corresponding to SEQ ID NO: 1 or that is complementary to a contiguous nucleotide sequence of a portion of the nucleotide sequence corresponding to SEQ ID NO: 1.
  • sense strand refers to a nucleotide sequence that corresponds to SEQ ID NO: 1 (or a contiguous nucleotide sequence of a portion of the nucleotide sequence corresponding to SEQ ID NO: 1 ) and thus is complementary to the antisense strand.
  • the terms "therapy,” and “treatment,” as used interchangeably herein, refer to an intervention performed with the intention of improving a recipient's status.
  • the improvement can be subjective or objective and is related to the amelioration of the symptoms associated with, preventing the development of, or altering the pathology of a disease, disorder or condition being treated.
  • therapy and treatment are used in the broadest sense, and include the prevention (prophylaxis), moderation, reduction, and curing of a disease, disorder or condition at various stages. Prevention of deterioration of a recipient's status is also encompassed by the term.
  • the disease, disorder or condition is prostate cancer, including benign prostate cancer, localized prostate cancer, indolent prostate cancer, mCRPC and other stage of prostate cancer.
  • subject or "patient,” as used herein, refers to a mammal in need of treatment.
  • an effective amount refers to an amount of a compound that produces a desired effect.
  • a population of cells may be contacted with an effective amount of a compound to study its effect in vitro (e.g., cell culture) or to produce a desired therapeutic effect ex vivo or in vitro.
  • An effective amount of a. compound may be used to produce a therapeutic effect in a subject, such as preventing or treating a target condition, alleviating symptoms associated with the condition, or producing a desired physiological effect.
  • the effective amount of a compound is a "therapeutically effective amount,” “therapeutically effective concentration” or “therapeutically effective dose.”
  • the precise effective amount or therapeutically effective amount is an amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject or population of cells. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication) or cells, the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration.
  • an effective or therapeutically effective amount may vary depending on whether the compound is administered alone or in combination with another compound, drug, therapy or other therapeutic method or modality.
  • One skilled in the clinical and pharmacological arts will be able to determine an effective amount or therapeutically effective amount through routine experimentation, namely by monitoring a cell's or subject's response to administration of a compound and adjusting the dosage accordingly.
  • Remington The Science and Practice of Pharmacy, 21 st Edition, Univ. of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, Pa., 2005, which is hereby incorporated by reference as if fully set forth herein.
  • in combination means in the course of treating the same disease in the same patient using two or more agents (including other siRNA or other ASO), drugs, treatment regimens, treatment modalities or a combination thereof, in any order.
  • Administration of a PCAT18 siRNA or an ASO "in combination with" one or more other anti-cancer therapeutics or chemotherapeutics is intended to include simultaneous (concurrent) administration and consecutive administration, as well as administration in a temporally spaced order of up to several days apart. Consecutive administration is intended to encompass administration of the other therapeutic(s) and the siRNA molecule(s) and/or the ASO molecule(s) to the subject in various orders.
  • Such combination treatment may also include more than a single administration of any one or more of the agents, drugs, treatment regimens or treatment modalities. Further, the administration of the two or more agents, drugs, treatment regimens, treatment modalities or a combination thereof may be by the same or different routes of administration.
  • a “biological sample” refers to any material, biological fluid, tissue, or cell obtained or otherwise derived from a subject including, but not limited to, blood (including whole blood, leukocytes, peripheral blood mononuclear cells, plasma, and serum), sputum, mucus, nasal aspirate, urine, semen, saliva, meningeal fluid, lymph fluid, milk, bronchial aspirate, a cellular extract, and cerebrospinal fluid. This also includes experimentally separated fractions of all of the preceding. For example, a blood sample can be fractionated into serum or into fractions containing particular types of blood cells, such as red blood cells or white blood cells (leukocytes).
  • a sample may be a combination of samples from an individual, such as a combination of a tissue and fluid sample.
  • a biological sample may also include materials containing homogenized solid material, such as from a stool sample, a tissue sample, or a tissue biopsy; or materials derived from a tissue culture or a cell culture.
  • Tissue may be normal tissue or cancerous tissue, such as a cancerous prostate tissue, a benign prostatic hyperplasia tissue, or normal prostate tissue.
  • PC AT 18 (SEQ ID NO:1 ; see Figure 5) is a long intergenic noncoding RNA at locus LOC728606, exhibiting high expression in a metastatic xenograft model (see Example 1 ). PCAT18 showed a similar magnitude of fold-change as the oncogenic IncRNAs H19 and PCGEM1 (see Figure 1 (B)).
  • Locus LOC728606 which encodes the intergenic IncRNA PCAT18, is flanked by AQP4 (Aquaporin-4) and KCTD1 (Potassium channel tetramerisation domain containing-1 ) loci and is part of the 18q11.2 genomic locus.
  • PCAT18 is a 2598 bp RNA containing 2 exons ( Figure 1(C)) and consists of the nucleotide sequence referenced as SEQ ID NO:1 ( Figure 5).
  • PCAT18 is significantly higher in normal prostate tissue than in normal tissues (see Figure 2(D)) and that it is over-expressed specifically in PCa as compared to 15 other neoplastic tissues ( Figure 2(C)), including, bladder cancer, brain and central nervous system cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, lymphoma, ovarian cancer, pancreatic cancer, and sarcoma.
  • PCAT18 is, therefore, prostate cancer-specific and prostate tissue-specific, which suggests its usefulness as a biomarker for disease detection, diagnosis and monitoring of PCa and for treatment of PCa, as indicated for other noncoding RNAs.
  • transcript variants may include, without limitation, a variant that is at least about 90% identical, at least about 91 % identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical to SEQ ID NO:1.
  • transcript variants that may be used in accordance with the methods described herein are not limited to those described above.
  • the transcript variants may include any additional variants of PCAT18 described herein and other IncRNAs that are transcribed from genomic locus LOC728606, as one skilled in the art would understand that many additional transcript variants related to PCAT18 may exist that have differential expression found in prostate cancer cells as compared to normal cells.
  • the transcript variant is capable of selectively hybridizing under stringent conditions to a portion of the genomic region at locus LOC728606.
  • Suitable stringent conditions include, for example, hybridization according to conventional hybridization procedures and washing conditions of 1 -3 x SSC, 0.1 -1 % SDS, 50-700C with a change of wash solution after about 5-30 minutes.
  • variations in stringency of hybridization conditions may be achieved by altering the time, temperature, and/or concentration of the solutions used for the hybridization and wash steps. Suitable conditions can also depend in part on the particular nucleotide sequences used.
  • modifications of the PCAT18 IncRNA may also be used as a biomarker for detecting, prognosing and monitoring cancer according to the methods and uses described herein.
  • Modifications of PCAT18 transcripts that may be detected and that may be indicative of PCa when used according to the methods and uses described herein may include, but are not limited to, single nucleotide polymorphisms (SNPs), DNA methylation or unmethylation, RNA methylation or unmethylation, and gene mutations or deletions.
  • SNPs single nucleotide polymorphisms
  • Such modifications may result in an alteration in the expression, formation, or conformation of the PCAT18 transcript in a cancerous or biological sample, as compared to a control, and may result in inhibition or impairment of a therapeutic agent targeting such PCAT18 transcript.
  • downstream targets of the PCAT18 transcript may be used as biomarkers for detecting, prognosing and monitoring cancer according to the methods described herein.
  • PC AT 18 transcript or "PCAT18” or “JUPITER” may be
  • PCAT 8 RNA comprising the nucleotide sequence referenced as SEQ ID NO: 1 , a variant transcript of PCAT18, as described above, comprising from about 90% to about 100%, or any amount therebetween, identity or sequence similarity with SEQ ID NO:1 , or a modification of either PCAT18 or a related transcript, or may be any other IncRNA that is transcribed from genomic locus LOC728606, which has increased expression in prostate cancer cells as compared to normal cells.
  • PCAT18 and/or one or more of the individual PCAT transcript variants, may be isolated from a biological sample (e.g., blood, serum, plasma, urine, saliva or prostate tissue) and the expression level of PCAT18 assessed in the biological sample to determine a diagnosis or prognosis of PCa and any stage of PCa.
  • a biological sample e.g., blood, serum, plasma, urine, saliva or prostate tissue
  • the expression of PCAT18 is tissue-specific (i.e., prostate tissue) and cancer-specific (PCa), with overexpression of PCAT18 in biological samples obtained from patients having PCa.
  • the PCAT18 transcript is associated with the presence or absence of primary PCa, metastatic PCa, including mCRPC, local or distant metastases, and the progression or aggressiveness of the PCa.
  • PCAT18 and other variants of PCAT18 and transcripts of the LOC728606 genomic locus may be used as biomarkers for diagnosing, prognosing, assessing risk and monitoring PCa. Further, such diagnoses, prognoses and assessments of risk of PCa based on expression levels of PCAT18 transcripts and related variants may be used to monitor a PCa patient's treatment and/or make clinical decisions regarding optimization of a PCa patient's treatment regimen.
  • the present invention relates to methods for diagnosis of a subject suspected of having prostate cancer, which involves assessing or determining PCAT18 expression levels in a biological sample obtained from the subject and comparing the expression level to a reference expression level.
  • the reference expression level may be obtained from the expression level of PCAT18 in samples obtained from normal individuals determined as not having PCa.
  • Such methods further include a step of diagnosing a subject as having PCa or identifying the subject as having PCa when the expression level of PCAT18 in the biological sample of the subject is greater than the reference expression level.
  • the subject may also be diagnosed as not having PCa or identified as not having PCa when the expression level of PCAT18 in the biological sample of the subject is not greater than a reference expression level.
  • the subject may be diagnosed with localized prostate cancer or a metastatic prostate cancer, including, without limitation, metastatic castration-resistant prostate cancer (mCRPC).
  • mCRPC metastatic castration-resistant prostate cancer
  • the diagnostic methods described herein may detect, determine, or recognize the presence or absence of PCa; prediction or diagnosis of metastasis or lack of metastasis, type or sub-type, or other classification or characteristic of PCa; whether a specimen is a benign lesion, such as benign prostatic hyperplasia (BPH), or a malignant tumor, or a combination thereof.
  • BPH benign prostatic hyperplasia
  • the expression level of PCAT18 in the subject's sample is at least about a 1.5 increase or fold- change over the reference expression level
  • the expression level of PCAT 18 may be at least about a 2 fold-change (as shown, for example, in Figure 3(B)), at least about a 2.3 fold-change (as shown, for example, in Figure 2(A)), at least about a 2.78 fold-change (as shown, for example, in Figure 2(C)), at least about a 3 fold-change, at least about a 3.5 fold-change, at least about a 4 fold-change, at least about a 4.5 fold-change, at least about a 5 fold-change, at least about a 5.5 fold-change (as shown in, for example, Figure 3(B)), at least about a 6 fold-change, at least about a 6.5 fold-change, at least about a 7 fold-change, at least about a 7.2 fold-change (a
  • the change in expression may be from about 1.5 to about 150, fold increase or fold-change, or any amount therebetween, over the reference expression level (as shown in Figure 3C: LNCap), or from about 1.5 to about 1000 fold increase or fold- change, or any amount therebetween, over the reference expression level (as shown in Figure 3C: C4-2).
  • the increase in expression of PCAT18 over the reference expression level may be about 1.5, 2, 4, 6, 8, 10, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 fold increase or fold- change, or any amount therebetween, over the reference expression level.
  • not greater than the reference expression level it is meant that the expression level of PCAT18 in the subject's sample is less than about a 1.4 increase or fold- change over the reference expression level, for example, the expression level of PCAT18 is less than about a 1.3 fold-change, less than about a 1.2 fold-change, less than about a 1.1 fold-change, less than about a 1 fold-change, less than about a 0.8 fold-change, less than about a 0.5 fold-change, less than about a 0.2 fold-change, less than about a 0.1 fold-change over the reference expression level.
  • the phrase "not greater than the reference expression level” may also include situations in which the expression level of PCAT 8 in the subject's sample is the same as or less than the reference expression level.
  • the present invention further relates to methods for determining the risk of a subject for developing prostate cancer.
  • Such methods comprise a step of assessing or determining the expression level of PCAT18 in a biological sample obtained from the subject and comparing the expression level to a reference expression level.
  • the reference expression level may be obtained from the expression level of PCAT18 in samples obtained from normal individuals determined as not having PCa.
  • Such methods further include a step of identifying the subject as having an increased risk of developing PCa when the expression level of PCAT18 in the biological sample of the subject is greater than the reference expression level.
  • the subject may also be identified as not having an increased risk of developing PCa when the expression level of PCAT18 in the biological sample of the subject is not greater than a reference expression level.
  • increase risk of developing PCa it is meant a greater than about a 10% chance of developing PCa as compared to a normal individual, for example, a greater than about a 15%, about a 20%, about a 25%, about a 30%, about a 35%, about a 40%, about a 45% or about a 50% chance of developing PCa as compared to a normal individual.
  • PCAT18 expression levels in a biological sample from a subject may also be used in the prognosis of a PCa patient (i.e., a subject having PCa), which involves assessing or determining PCAT18 expression levels in a biological sample obtained from the subject and comparing the expression level to a threshold level.
  • Such methods described herein may, therefore, include a step of determining a prognosis for a subject having PCa when an expression level of PCAT18 is greater than, less than or within the threshold level.
  • the prognosis may refer to a prediction of a future course of PCa in a subject who has the disease or condition (e.g., predicting disease outcome, such as, but not limited to, predicting patient survival), and may also encompass the evaluation of the response or outcome of the disease in the individual after administering a treatment or therapy to the individual, and may refer to a prediction of an increased or reduced risk of PCa relapse.
  • the prognosis may be a poor prognosis or a good prognosis, as measured by a decreased length of survival or a prolonged (or increased) length of survival, respectively.
  • the prognosis may be a poor prognosis if the expression level of PCAT18 in the subject's biological sample is greater than the threshold level; that is, if the expression level of PCAT18 in the subject's biological sample is greater than about a 4 fold-change over the reference expression level.
  • the prognosis may be good if the expression level of PCAT18 in the subject's biological sample is within the threshold level; that is, if the expression level of PCAT18 is between about a 1.1 fold-change and about a 4 fold-change over the reference expression level.
  • the prognosis may be even better if the expression level of PCAT18 in the subject's biological sample is less than the threshold level.
  • the methods described herein may also be used to differentiate between an early stage cancer (i.e., primary tumor); or a metastasized PCa when the expression level is significantly different than threshold level.
  • a method for determining a risk of metastatic spread of (i.e. risk of metatsis in other organs or parts of the body that can be determined using standard tests) PCa in a subject diagnosed with PCa is provided herein.
  • Such a method involves assessing or determining the expression level of PCAT18 in a biological sample obtained from the subject diagnosed with PCa and comparing the expression level to a threshold level. The subject is identified as having an increased risk of metastatic spread when the expression level of PCAT18 in the subject's biological sample is significantly greater than the threshold level.
  • the expression level of the PCAT18 in the subject's biological level is at least about a 6 fold-change over a reference expression level, and may be about a 7 fold- change, about an 8-fold-change, about a 9 fold-change, about a 10 fold-change, about an 11 fold-change, about a 12 fold-change, about a 13 fold-change, about a 14 fold-change, about a 15 fold-change, about a 16 fold-change, about a 17 fold-change, about an 18 fold-change, about a 19 fold-change, about a 20 fold-change, about a 30 fold-change, about a 40 fold- change, about a 50 fold-change, about a 60 fold-change, about a 70 fold-change, about an 80 fold-change, about a 90 fold-change, about a 100 fold-change, about a 200 fold-change, about a 300 fold-change, about a 400 fold-change, about a 500 fold-change, about
  • the increased risk of metastatic spread includes, for example, without limitation, an increased risk of a locoregional metastasis, a distant metastasis or an increased risk of progression to a more clinically aggressive PCa, including, mCRPC.
  • the present invention further relates to methods for monitoring a treatment administered to a patient diagnosed with PCa and involves analyzing the expression level of PCAT18 at two different timepoints, such as prior to administration of treatment and after administration of treatment.
  • the method comprises obtaining a baseline level of expression of PCAT18 in a biological sample obtained from the subject. This baseline level is obtained prior to administration of a treatment, or prior to a second timepoint at which an expression level of PCAT18 will be determined.
  • the method then comprises the step of administering the treatment for a treatment period and then determining or assessing the expression level at a second timepoint from a second biological sample obtained from the subject.
  • a comparison of the expression level at the second timepoint to the baseline level will identify whether the patient has responded poorly to the treatment or whether the patient has had a good response to the treatment.
  • the second timepoint may also be after a certain period of time has elapsed from obtaining the first biological sample from the subject, without a treatment step in between. This may be the case if the method comprises a step of determining whether a treatment course, treatment regimen or treatment modality should be started, for example, if the patient's PCa was in remission and determining whether there has been a relapse in the patient, or if the patient's disease has progressed to mCRPC and determining whether surgery or hormonal therapy should be administered.
  • the methods described herein may also include monitoring or assessing the progression of PCa in a subject; monitoring or assessing a response to treatment in a subject having PCa; monitoring or assessing a metastatic spread of PCa in a subject; monitoring or assessing a remission state or a recurrence of PCa in a subject or a combination thereof.
  • Such monitoring or assessing may include an individual's response to a therapy, such as, for example, predicting whether an individual is likely to respond favorably to a therapeutic agent, is unlikely to respond to a therapeutic agent, or will likely experience toxic or other undesirable side effects as a result of being administered a therapeutic agent; selecting a therapeutic agent for administration to an individual, or monitoring or determining an individual's response to a therapy that has been administered to the individual.
  • An expression level of PCAT18 in a subject or a reference expression level used in the methods for diagnosis, prognosis, monitoring, treating, or assessing risk of developing PCa or progression to metastatic risk, as described herein, may be measured, quantified and/or detected by any suitable RNA detection, quantification or sequencing methods known in the art, including, but not limited to, quantitative PCR (QPCR) or quantitative/gel-based electrophoresis PCR, .reverse transcriptase-polymerase chain reaction (RT-PCR) methods, microarray, serial analysis of gene expression (SAGE), next-generation RNA sequencing (e.g., deep sequencing, whole transcriptome sequencing, exome sequencing), gene expression analysis by massively parallel signature sequencing (MPSS), immune-derived colorimetric assays, in situ hybridization (ISH) formulations (colorimetric/radiometric) that allow histopathology analysis, mass spectrometry (MS) methods, RNA pull-down and chromatin isolation by RNA purification (ChiRP), and proteomics-based
  • the method of measuring the expression level of PCAT18 may also include non- PCR-based molecular amplification methods for detection.
  • a combination of the above methods for assessing the expression level or reference expression level is also contemplated.
  • a diagnosis or prognosis of PCa based on the methods described herein may be used to optimize or select a treatment regimen for a subject diagnosed with PCa.
  • a method for diagnosing or prognosing PCa may be performed as described above.
  • a subject that is diagnosed with primary PCa based on an expression level of PCAT18 or a related variant may be treated according to FDA approved protocols and standards known in the art for a particular therapeutic agent for primary PCa.
  • a diagnosis of primary PCa may be treated using surgery, such as, but not limited to, radical prostatectomy, and/or primary PCa may be treated using a "wait and see approach" before or after surgery, since such a diagnosis indicates that metastasis of the primary PCa has not occurred.
  • primary PCa may be treated using a therapeutic or pharmaceutical agent that targets PCAT18 and/or one or more related variants, and inhibits or silences the expression of PCAT18, as described below.
  • monitoring of the PCa and progression of the PCa to a more clinically aggressive PCa is performed by periodically assessing the expression levels of PCAT18 and/or any related variants in a patient's biological sample, such as blood, plasma, urine or prostate tissue.
  • a diagnosis of a subject that is diagnosed with metastatic PCa based on an expression level of PCAT18 or a related variant may be treated more aggressively according to FDA approved protocols and standards known in the art for metastatic PCa.
  • a diagnosis of a more aggressive PCa may be treated using surgery, such as, but not limited to, radical prostatectomy, if such a surgery is deemed acceptable.
  • a more aggressive PCa may be treated using a therapeutic or pharmaceutical agent that targets PCAT18 and/or one or more related variants, and inhibits or silences the expression of PCAT18, as described below.
  • monitoring of the PCa is performed by periodically assessing the expression levels of PCAT18 and/or any related variants in a patient's biological sample, such as blood, plasma, urine or prostate tissue.
  • the present invention also relates to targeting PCAT18, including but not limited to additional transcript variants of PCAT18, modifications of PCAT18 and related variants, and other IncRNAs that may be transcribed from genomic locus LOC728606, using an inhibiting agent or therapeutic targeting strategy, such as antisense oligonucleotides, RNA interference (RNAi), esiRNA, shRNA, miRNA, decoys, RNA aptamers, small molecule inhibitors, RNA/DNA-binding proteins/peptides or other compounds with different formulations to inhibit one or more physiological actions effected by PCAT18 and to thereby treat PCa.
  • an inhibiting agent or therapeutic targeting strategy such as antisense oligonucleotides, RNA interference (RNAi), esiRNA, shRNA, miRNA, decoys, RNA aptamers, small molecule inhibitors, RNA/DNA-binding proteins/peptides or other compounds with different formulations to inhibit one or more physiological actions effected by PCAT18 and
  • Such therapeutic targeting strategies may be used to develop a therapeutic agent or pharmaceutical compositions that target PCAT18 and/or one or more related variants for treating PCa.
  • Treatment of PCa may include administering to a subject having PCa a therapeutically effective amount of a therapeutic agent, such as an inhibiting agent of PCAT18 or a pharmaceutical composition, as described herein.
  • a therapeutic agent such as an inhibiting agent of PCAT18 or a pharmaceutical composition, as described herein.
  • the inhibiting agent of PCAT18 may be an antisense oligonucleotide, RNA interference (RNAi), siRNA, esiRNA, shRNA, miRNA, decoys, RNA aptamers, small molecule inhibitors, RNA DNA-binding proteins/peptides, or a combination thereof.
  • RNAi RNA interference
  • siRNA molecules esiRNA, shRNA, miRNA, decoys, RNA aptamers, small molecule inhibitors, RNA DNA-binding proteins/peptides, or a combination thereof.
  • the present invention provides for methods of treating PCa in a subject diagnosed with PCa using small interfering RNA (siRNA) molecules against PCAT18.
  • siRNA molecules targeted to PCAT 8 have been found to decrease proliferation of cancer cells when used as a single agent.
  • siRNAI which comprises a nucleotide sequence corresponding to SEQ ID NO:22
  • siRNA2 which comprises a nucleotide sequence corresponding to SEQ ID NO:23
  • siRNAI and siRNA2 silencing of PCAT18 elicited significant and stable growth inhibition in a human prostate cancer cell line (C4-2) (see Figures 3(D) and 3(E)).
  • siRNAs used in the present invention are targeted to a PCAT18 gene, or the genomic region at locus LOC728606, and are capable of silencing or inhibiting the expression of PC AT 18 RNA.
  • siRNAs targeted to a PCAT18 gene or locus LOC728606 comprise a specific antisense sequence that is complementary to a portion of the noncoding RNA transcribed from the target gene (i.e., the target RNA) and can be double-stranded (i.e. composed of an antisense strand, comprising the specific antisense sequence, and a complementary sense strand) or single-stranded (i.e. composed of an antisense strand, comprising the specific antisense sequence, only) as described in more detail below.
  • Short- hairpin siRNA against PCAT18 are also included in the present invention.
  • siRNA Short- hairpin siRNA
  • the specificity of siRNA molecules is determined by the binding of the antisense strand of the molecule to its target RNA.
  • Effective siRNA molecules are generally from 14 to 100 base pairs in length, or any length therebewteen to prevent them from triggering non-specific RNA interference pathways in the cell via the interferon response, although longer siRNA can also be effective.
  • the siRNA molecules contemplated by the present invention may be 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 base pairs in length or any number of base pairs therebetween in length.
  • siRNA molecules Design and construction of siRNA molecules is known in the art (see, for example, Elbashir, et al., Nature, 411 :494-498 (2001 ); Bitko and Barik, BMC Microbiol., 1 :34 (2001 )].
  • the target RNA is a noncoding RNA transcribed from the PCAT18 gene or the genomic region at locus LOC728606, including, without limitation, the nucleotide sequence corresponding to SEQ ID NO: 1 (shown in Figure 5). Therefore, in an embodiment, the target RNA for the PCAT18 siRNA is PCAT 8 RNA corresponding to the nucleotide sequence as set forth in SEQ ID NO: 1.
  • the siRNA may comprise a sequence that is complementary to a target sequence within SEQ ID NO: 1.
  • Suitable target sequences within the target RNA are selected using one or more of several criteria known in the art (see for example, Elbashir, S. M., et al. (2001 ) Nature 41 1 , 494-498; Elbashir, S. M., et al. (2002) Methods 26, 199-213; Elbashir, S. M., et al. (2001 ) Genes Dev. 15, 188-200; Elbashir, S. M., et al. (2001 ) EMBO J. 20, 6877- 6888; and Zamore, P.D., et al. (2000) Cell 101 , 25-33).
  • Target RNA sequences within the target RNA are typically between about 14 and about 50 nucleotides in length, or any length therebewteen, but may be longer in length, for example, the target RNA sequence may be about 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 base pairs in length, or any number of base pairs therebetween in length.
  • the target RNA sequence can be selected from various regions within the PCAT18 RNA.
  • siRNAI comprises an antisense sequence SEQ ID NO:22 which is complementary to a target sequence within SEQ ID NO: 1
  • siRNA2 comprises an antisense sequence SEQ ID NO:23, which is complementary to a target sequence within SEQ ID NO: 1.
  • siRNA molecules that comprise a nucleotide sequence complementary to all or a portion of the target RNA sequence, i.e. an antisense sequence, can be designed and prepared.
  • the siRNA molecule can be double stranded (i.e. a dsRNA molecule comprising an antisense strand and a complementary sense strand) or single- stranded (i.e. a ssRNA molecule comprising just an antisense strand).
  • the siRNA molecules can comprise a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense strands.
  • Double-stranded siRNA may comprise RNA strands that are the same length or different lengths.
  • the siRNA is a double-stranded siRNA.
  • the siRNA is a double-stranded siRNA wherein both RNA strands are the same length.
  • Double-stranded siRNA molecules can also be assembled from a single oligonucleotide in a stem-loop structure, wherein self-complementary sense and antisense regions of the siRNA molecule are linked by means of a nucleic acid based or non-nucleic acid-based linker(s), as well as circular single-stranded RNA having two or more loop structures and a stem comprising self-complementary sense and antisense strands, wherein the circular RNA can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNA interference.
  • Small hairpin RNA (shRNA) molecules thus are also contemplated by the present invention. These molecules comprise a specific antisense sequence in addition to the reverse complement (sense) sequence, typically separated by a spacer or loop sequence. Cleavage of the spacer or loop provides a single-stranded RNA molecule and its reverse complement, such that they may anneal to form a dsRNA molecule (optionally with additional processing steps that may result in addition or removal of one, two, three or more nucleotides from the 3' end and/or the 5' end of either or both strands).
  • the spacer can be of a sufficient length to permit the antisense and sense sequences to anneal and form a double-stranded structure (or stem) prior to cleavage of the spacer (and, optionally, subsequent processing steps that may result in addition or removal of one, two, three, four, or more nucleotides from the 3' end and/or the 5' end of either or both strands).
  • the spacer sequence is typically an unrelated nucleotide sequence that is situated between two complementary nucleotide sequence regions which, when annealed into a double-stranded nucleic acid, comprise a shRNA (see, for example, Brummelkamp et al., 2002 Science 296:550; Paddison et al., 2002 Genes Develop.
  • the spacer sequence generally comprises between about 3 and about 00 nucleotides.
  • Single-stranded siRNA molecules are generally single-stranded RNA molecules with little or no secondary structure.
  • the overall length of the siRNA molecules can vary from about 14 to about
  • siRNAs may be siRNAI and siRNA2, as described above, corresponding to SEQ ID NO: 22 and SEQ ID NO: 23, respectively, which are each 36 oligonucleotides in length.
  • the siRNA molecule is a shRNA molecule or circular siRNA molecule between about 35 and about 100 nucleotides in length. In a further embodiment, the siRNA molecule is a shRNA molecule between about 40 to about 60 nucleotides in length.
  • the siRNA molecule comprises an antisense strand that includes a specific antisense sequence complementary to all or a portion of a target RNA sequence, such as, the PCAT18 noncoding RNA.
  • a target RNA sequence such as, the PCAT18 noncoding RNA.
  • the antisense strand of the siRNA molecules may comprise a specific antisense sequence together with nucleotide sequences at the 5' and/or 3' termini that are not complementary to the target sequence.
  • Such non- complementary nucleotides may provide additional functionality to the siRNA molecule. For example, they may provide a restriction enzyme recognition sequence or a "tag" that facilitates detection, isolation or purification. Alternatively, the additional nucleotides may provide a self-complementary sequence that allows the siRNA to adopt a hairpin configuration. Such configurations are useful when the siRNA molecule is a shRNA molecule, as described above.
  • the specific antisense sequence comprised by the siRNA molecule can be identical or substantially identical to the complement of the target RNA sequence.
  • the specific antisense sequence comprised by the siRNA molecule can be identical or substantially identical to the complement of the PCAT18 RNA sequence, that is, the complement of a contiguous portion of the RNA sequence corresponding to SEQ ID NO:1.
  • the specific antisense sequence comprised by the siRNA molecule is at least about 75% identical to the complement of a contiguous portion of the RNA sequence corresponding to SEQ ID NO:1.
  • the specific antisense sequence comprised by the siRNA molecule is at least about 90% identical to the complement of a contiguous portion of the RNA sequence corresponding to SEQ ID NO:1. In a further embodiment, the specific antisense sequence comprised by the siRNA molecule is at least about 95% identical to the complement of a contiguous portion of the RNA sequence corresponding to SEQ ID NO:1. In another embodiment, the specific antisense sequence comprised by the siRNA molecule is at least about 98% identical to the complement of a contiguous portion of the RNA sequence corresponding to SEQ ID NO:1.
  • the siRNA molecules comprise a specific antisense sequence that is capable of selectively hybridizing under stringent conditions to a portion of a naturally occurring target RNA, such as PCAT18 RNA.
  • Suitable stringent conditions include, for example, hybridization according to conventional hybridization procedures and washing conditions of 1-3 x SSC, 0.1-1% SDS, 50-700C with a change of wash solution after about 5-30 minutes.
  • siRNA molecules can be prepared using several methods known in the art, such as chemical synthesis, in vitro transcription, the use of siRNA expression vectors, and any other conventional techniques known in the art. For example, general methods of RNA synthesis and use of appropriate protecting groups is well known in the art (see, for example, Scaringe, S. A., et al., J. Am. Chem.
  • modified siRNA molecules such as phosphorothioated and alkylated derivatives, can also be readily prepared by similar methods.
  • siRNA molecules Various methods of testing the efficacy of the siRNA molecules are known in the art and may be employed to test the efficacy of the PCAT18 siRNA molecules, including siRNAI and siRNA2.
  • the present invention also provides for methods of treating PCa in a subject diagnosed with PCa using antisense oligonucleotides (ASOs).
  • ASOs targeted to PCAT 8 have been found to decrease proliferation of cancer cells when used as a single agent.
  • AS02 which comprises a nucleotide sequence corresponding to SEQ ID No:24
  • AS07 which comprises a nucleotide sequence corresponding to SEQ ID NO:25 both independently silence PCAT18.
  • AS02 and AS07 inhibition of PCAT18 elicited significant knockdown of PCAT18 expression in a human prostate cancer cell line (C4-2) as compared to an antisense nucleotide (NC) with no known specific target in human or mouse genome (see Figure 10).
  • ASOs used in the present invention are targeted to PCAT18 RNA, or any other additional RNA transcribed from the genomic region at locus LOC728606.
  • the ASOs of the present invention are effective in reducing the amount of expression of PCAT18 RNA in vivo.
  • ASOs targeted to the PCAT18 RNA or other transcripts derived from locus LOC728606 comprise a specific antisense sequence that is complementary to a portion of the noncoding RNA transcribed from the target gene (i.e., the target RNA) and can be either DNA, RNA or a chemical analogue.
  • ASOs are generally single-stranded (i.e.
  • Suitable ASOs have a length of from about 12 to about 35 oligonucleotides and any amount therebewteen, and have sequence specificity (i.e., are complementary) to the PCAT18 noncoding RNA sequence.
  • the ASOs of the present invention may comprise more than about 35 oligonucleotides, for example, about 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 oligonucleotides in length, or any number of oligonucleotides therebetween.
  • Exemplary ASOs comprise a nucleotide sequence complementary to a contiguous portion of the nucleotide sequence (i.e. a target sequence) corresponding to SEQ ID NO: 1.
  • the contiguous portion of the nucleotide sequence may be between about 12 to about 250 oligonucleotides in length, or any number of oligonucleotides in length therebetween.
  • the ASOs may be AS02 and AS07, as described above, corresponding to SEQ ID NO: 24 and SEQ ID NO: 25, respectively, which are each 20 oligonucleotides in length.
  • the specific antisense sequence comprised by an ASO of the present invention can be identical or substantially identical to the complement of the target RNA sequence.
  • the specific antisense sequence comprised by the ASO molecule can be identical or substantially identical to the complement of the PCAT18 RNA sequence, that is, the complement of a contiguous portion of the RNA sequence corresponding to SEQ ID NO: 1.
  • the specific antisense sequence comprised by the ASO molecule is at least about 75% identical to the complement of a contiguous portion of the RNA sequence corresponding to SEQ ID NO: 1.
  • the specific antisense sequence comprised by the ASO molecule is at least about 90% identical to the complement of a contiguous portion of the RNA sequence corresponding to SEQ ID NO: 1.
  • the specific antisense sequence comprised by the ASO molecule is at least about 95% identical to the complement of a contiguous portion of the RNA sequence corresponding to SEQ ID NO: 1. In another embodiment, the specific antisense sequence comprised by the ASO molecule is at least about 98% identical to the complement of a contiguous portion of the RNA sequence corresponding to SEQ ID NO: 1.
  • Methods of determining sequence identity are known in the art and can be determined, for example, by using the BLASTN program of the University of Wisconsin Computer Group (GCG) software or provided on the NCBI website.
  • the ASO molecules comprise a specific antisense sequence that is capable of selectively hybridizing under stringent conditions to a portion of a naturally occurring target RNA, such as PCAT18 RNA or any other RNA transcribed from the genomic region at locus LOC728606.
  • Suitable stringent conditions include, for example, hybridization according to conventional hybridization procedures and washing conditions of 1-3 x SSC, 0.1-1 % SDS, 50-700C with a change of wash solution after about 5-30 minutes.
  • variations in stringency of hybridization conditions may be achieved by altering the time, temperature, and/or concentration of the solutions used for the hybridization and wash steps.
  • Suitable conditions can also depend in part on the particular nucleotide sequences used, for example the portion of the antisense sequence corresponding to SEQ ID NO: 1.
  • the oligonucleotides employed as ASOs in the present invention may be modified to increase the stability of the ASOs in vivo.
  • the ASOs may be employed as phosphorothioate derivatives (replacement of a non-bridging phosphoryl oxygen atoms with a sulfur atom) which have increased resistance to nuclease digestion (as done with AS02 and AS07).
  • MOE modification ISIS backbone is also effective.
  • the ASOs used in the present invention may be prepared according to any of the methods that are well known to those of ordinary skill in the art.
  • the ASOs may be prepared by solid phase synthesis. See, Goodchild, J., Bioconjugate Chemistry,! :165-167 (1990), for a review of the chemical synthesis of oligonucleotides.
  • the ASOs can be obtained from a number of companies which specialize in the custom synthesis of oligonucleotides.
  • Administration of the therapeutic agents described herein can be carried out using the various mechanisms known in the art, including naked administration and administration in pharmaceutically acceptable lipid carriers.
  • lipid carriers for ASO delivery are disclosed in U.S. Pat. Nos. 5,855,91 1 and 5,417,978 which are incorporated herein by reference.
  • the carrier may also be any one of a number of sterols including cholesterol, cholate and deoxycholic acid.
  • the therapeutic agents describe herein, including the ASOs and siRNA molecules may be administered by intravenous, intraperitoneal, subcutaneous or oral routes, or direct local tumor injection.
  • Suitable formulations for parenteral administration include aqueous solutions of the therapeutic agents in water-soluble form, for example, water-soluble salts.
  • suspensions of the active compounds as appropriate oily injection suspensions may be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran.
  • the suspension may also contain stabilizers.
  • a therapeutic agent may be co-administered with an agent which enhances the uptake of the therapeutic agent by the cells.
  • a therapeutic agent may be combined with a lipophilic cationic compound which may be in the form of liposomes.
  • liposomes to introduce nucleotides into cells is taught, for example, in U.S. Patent Nos. 4,897,355 and 4,394,448, the disclosures of which are incorporated by reference in their entirety. See also U.S. Patent Nos.
  • the therapeutic agents described herein may be conjugated to a peptide that is ingested by cells.
  • useful peptides include peptide hormones, antigens or antibodies, and peptide toxins. By choosing a peptide that is selectively taken up by the cancerous prostate cells, specific delivery of the therapeutic agent may be effected.
  • the amount of a therapeutic agent administered in the present methods describe herein is one effective to reduce the amount of PCAT18 expression.
  • the therapeutic agents described herein may also be administered as part of a pharmaceutical composition or preparation containing suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the therapeutic agents into preparations which can be used pharmaceutically.
  • suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the therapeutic agents into preparations which can be used pharmaceutically.
  • the present invention contemplates pharmaceutical compositions comprising a therapeutic agent effective to reduce the amount of PCAT18 in cancerous prostate cells exposed to the therapeutic agent, and a pharmaceutically acceptable carrier.
  • the therapeutic agent may be an inhibiting agent of PCAT18, such as, for example, antisense oligonucleotides, RNA interference (RNAi), esiRNA, shRNA, miRNA, decoys, RNA aptamers, small molecule inhibitors, RNA/DNA-binding proteins/peptides or other compounds which inhibit the expression of PCAT18.
  • the pharmaceutical composition may comprise one or more than one therapeutic agent, and a pharmaceutically acceptable carrier.
  • compositions used in the present invention include all compositions wherein the one or more than one therapeutic agent is contained in an amount which is effective to achieve inhibition of expression of PCAT18. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art.
  • the present invention further contemplates a method of treating PCa in a subject comprising the administration of a therapeutically effective amount of a PCAT18 siRNA in combination with any other treatment, agent, drug, regimen or therapy, including without limitation, administration of ASOs, hormonal therapy, surgery, radiation therapy, chemotherapy, biologic therapy, bisphosphonate therapy, cryosurgery, high-intensity focused ultrasound, and proton beam radiation therapy.
  • a method of treating PCa in a subject diagnosed with PCa may comprise administering a therapeutically effective amount of an ASO in combination with any other treatment or therapy, including without limitation, administration of PCAT18 siRNA molecules, hormonal therapy, surgery, radiation therapy, chemotherapy, biologic therapy, bisphosphonate therapy, cryosurgery, high-intensity focused ultrasound, and proton beam radiation therapy.
  • PCa biopsy specimens were collected at the BC Cancer Agency with the patient's written informed consent. The protocol for this procedure was approved by the University of British Columbia (UBC) Research Ethics Board (REB). NOD/SCID mice used for this study were bred and maintained at the British Columbia Cancer Research Centre Animal Facility (Vancouver, Canada). All experimental protocols were approved by the University of British Columbia Animal Care Committee. Transplantable PCa tissue xenograft lines were established and maintained using subrenal capsule grafting as previously described (10).
  • RNA Sequencing [00145] Total RNA was extracted from non-metastatic LTL-313B and metastatic LTL-
  • RMS root mean square
  • Mapped transcripts were annotated using the gene cards database (www.qenecards.org). Genes were categorized as "protein coding” and "non-coding” based on their functional annotation. Among non-coding sequences rRNAs, tRNAs, miRNAs snoRNAs and other known classes of RNAs were excluded from further analysis. LncRNAs were defined as all non-coding sequences longer than 200 bp and not belonging to other RNA categories. Based on those filtering criteria, 1653 IncRNAs expressed in PCa xenografts were identified.
  • LOC728606 expression was also queried in Oncomine (www.oncomine.com) GEO (www.ncbi.nlm.nih.gov/geo/) and Cbio portal (www.cbioportal.org) gene expression databases. Analysis was restricted to PCa and prostate-derived samples.
  • RNAs were analyzed through the cBio cancer genomic portal (12), which includes clinico-pathological and gene expression information from 29 normal prostate and 131 primary PCa samples (13). Gene expression data were downloaded from the portal as log2 whole transcript normalized RNA expression values (Affymetrix Human Exon 1.0 ST arrays). To further characterize LOC728606 (JUPITER), expression patterns of JUPITER were analyzed in Oncomine (www.oncomine.com) and Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) databases, which include large collections of microarray data from human samples.
  • Oncomine www.oncomine.com
  • GEO Gene Expression Omnibus
  • Prostate tissue samples Samples from patients with benign prostatic hyperplasia (BPH) or PCa were collected at the Stephanshorn Clinic in St. Gallen Switzerland, after study protocol approval by the local ethical committee. Resected specimens were immediately transferred on ice to the Institute for Pathology of the Kantons Hospital, St.Gallen for examination. Small tissue samples from macroscopically visible tumor and non-tumor prostate tissue were dissected, snap frozen in liquid nitrogen and cryo- preserved at -80 °C. These samples were cut in a cryo-microtome and a slide of each probe was stained with hematoxylin-eosin for histological verification. RNA was isolated from frozen materials using the TRI-reagent (Ambion) method according to the manufacturer's guidelines.
  • the cDNA was synthesized from 1 pg of total RNA using Superscript II RNase H-reverse transcriptase (Invitrogen).
  • Plasma Samples Upon study protocol approval by UBC REB, and after obtaining written informed consent from study participants, blood samples and clinico- pathological data were collected at the British Columbia Cancer Agency (BCCA), Vancouver Centre. Three cohorts were evaluated: 25 individuals with no clinical sign of neoplasm; 25 PCa patients with treatment-naive localized disease (Localized PCa); 25 patients with a clinically confirmed metastatic PCa and a progressive disease despite castration therapy (mCRPC). Samples were processed as previously described (4) for plasma separation, RNA extraction and retrotranscription.
  • BCCA British Columbia Cancer Agency
  • Table 1 Clinical-pathological characteristics of enrolled patients.
  • Risk groups in Table 1 are defined based on pre-prostatectomy serum PSA value, T stage and Gleason Grade, as recommended by the Genito-Urinary Radiation Oncologists of Canada (2). PCa diagnosis was confirmed by pathological examination of tumor biopsies for each enrolled patient. Localized PCa cases were defined as those with no pathological evidence of lymph node dissemination and no clinical evidence of metastatic diffusion. PSA measurement and RNA extraction were performed on samples collected before prostatectomy and on treatment-naive patients. Metastatic cases were defined as those having clinical or pathological evidence of cancer dissemination to any of the following: lymph nodes, bones or soft tissues (lung, brain, spine, testis).
  • RNA extracted from xenografts as described above, was retrotranscribed using QuantiTect Reverse Transcription kit (Qiagen) following manufacturer's instructions. RNA extraction and retrotranscription for clinical samples are described above.
  • Biosystem Non-coding RNA assay Hs03669364_m1 was employed, which is specific for LOC728606 (PCAT18) and spans the exon1-exon2 boundary.
  • QPCR was performed according to manufacturer's instructions on the ABIPrism 7900HT (Applied Biosystems).
  • the 2 " ⁇ metnoc j was usec
  • TaqMan qPCR was also performed to quantify the sub-cellular localization of PCAT18. GAPDH and MALAT1 (Hs00273907_s1 ). Total, cytoplasmic and nuclear RNA was extracted and purified using the Ambion PARIS kit (Life Technologies), following manufacturer's instruction.
  • Prostate cancer- and benign prostatic hyperplasia-derived cell lines were maintained in 10% fetal bovine serum (GIBCO, Life Technologies ) and RPMI 1640 growth medium (GIBCO, Life Technologies).
  • MTT assay was performed on LNCaP, C4-2 and BPH cells treated with NC or PCAT18-targeting siRNAs (both at 2nM concentration) on days 1-3-5 post-transfection, as previously described (Watahiki A, et al. MicroRNAs associated with metastatic prostate cancer. PloS one. 2011 ; 6(9):e24950).
  • the wound healing assay was performed in triplicate on C4-2 cells as previously described (Decker KF, et al. Persistent androgen receptor-mediated transcription in castration-resistant prostate cancer under androgen-deprived conditions. Nucleic acids research. 2012; 40(21 ): 10765-10779). Transfection protocols were identical to those described above. 12 hours post-transfection, a 'wound' was produced using a P20 pipette tip. Pictures were taken at marked spots 0-6-24-48h post-wounding, using a Zeiss Axiovert 40 CFL inverted microscope connected to Axiovision 4.7 software.
  • Invasion assay was performed in triplicate on C4-2 cells using BD BioCoatTM BD MatrigelTM Invasion Chambers (24-well plates) and following manufacturer's instructions. Transfection was performed on day 0, as described above. After 12 hours, cells were plated in the invasion chambers. 16 hours post-plating, we followed a previously described method for analysis and quantification of invading cells (Crea F, et al. Pharmacologic disruption of Polycomb Repressive Complex 2 inhibits tumorigenicity and tumor progression in prostate cancer. Molecular cancer. 201 1 ; 10:40).
  • Antisense Oligonucleotide Knockdown C4-2 cells were treated with 160nM of PCAT18-targeting antisense oligonucleotides (AS02 and AS07), or antisense oligonucleotide (NC) with no known specific target in human or mouse genome.
  • ASO sequences AS02 and AS07 are detailed in Figures 9(B) and 9(C), respectively.
  • the antisense oligonucleotide (NC) is detailed in Figure 9(A).
  • the antisense oligonucleotides were purchased from Integrated DNA Technologies.
  • the ' * ' in the sequences represent phosphorothioate backbone.
  • oligonucleotides were re-suspended in 1X TE buffer as per manufacturer's instructions.
  • ASO transfections were performed following manufacturer's instructions using Oligofectamine (Life Technologies) as the transfecting reagent.
  • Gene quantification via quantitative PCR was performed as described above.
  • LTL-313B cells showed little local invasion and no distant metastasis while LTL-313H xenografts (metastatic) showed invasion into the mouse host kidney and distant metastases were detectable in the hosts' lungs 3 months after engraftment (Fig. 1 A).
  • RNA Sequencing was performed on paired metastatic/non-metastatic PCa orthotopic xenografts derived from clinical specimens. The most differentially expressed IncRNA was further analyzed in clinical samples and publically available databases.
  • genes were categorized as "protein coding” and "non- coding” based on their functional annotation. Among non-coding sequences rRNAs, tRNAs, miRNAs and other known classes of RNAs were excluded from further analysis. LncRNAs were defined as all non-coding sequences longer than 200 bp and not belonging to other RNA categories. Based on those filtering criteria, 1668 IncRNAs expressed in PCa xenografts were identified.
  • IncRNAs Up-regulated in Metastatic (313H) vs. Localized (313B)
  • the transcript with highest expression in the metastatic xenograft was LOC728606, a previously uncharacterized gene. This transcript showed a similar magnitude of fold-change with 2 previously known oncogenic IncRNAs (FigI B). LOC728606, flanked by AQP4 (Aquaporin-4) and KCTD1 (Potassium channel tetramerisation domain containing-1 ) loci, is transcribed to generate a 2598 bp RNA containing 2 exons (FigI C), and is classified as a "long intergenic non-coding RNA" based on Ensembl algorithm (www.ensembl.org).
  • LOC728606 was investigated in publically available databases, for example, the OncomineTM ( Figure 2A) and cBio ( Figure 2B) databases. LOC728606 expression profiles were mined on Oncomine and Gene Expression Omnibus (GEO) databases, which include large collections of microarray data from human samples. LOC728606 is significantly up-regulated in PCa vs. normal tissue in both the OncomineTM ( Figure 2A) and cBio ( Figure 2B) databases. The data from the OncomineTM analysis is summarized below in Table 7. [00183] Table 7. Summary of all OncomineTM Outputs for LOC728606 in PCa, with p value >0.01 and/or fold change ⁇ 2.
  • this LOC728606 gene is significantly over-expressed in normal prostate compared to 1 1 other benign tissues (Fig2C) and in PCa compared to 15 other neoplastic tissues (Fig2D). Based on its chromosomal location and prostate cancer- specificity, this new gene was originally called JUPITER (Just Uncoding, Prostate-specific, Intergenic Transcript located on Eighteen chromosome Region q1 1.2). The HUGO Gene Nomenclature Committee has officially named this noncoding RNA PCAT18.
  • JUPITER the expression levels of JUPITER were analyzed in human prostate tissue and plasma samples using quantitative PCR (QPCR). JUPITER was highly up-regulated (8.8-1 1.1 fold, p ⁇ 0.001 ) in both low-Gleason and high-Gleason PCa samples, compared to benign prostatic hyperplasia (BPH) (Fig 3A). Therefore, JUPITER up- regulation is not a mere function of prostate cells' hyper-proliferation.
  • LOC728606 could be detected in plasma samples, and if it could be exploited as a biomarker for disease detection and monitoring.
  • JUPITER expression (measured by QPCR) was also significantly elevated in 5 well-known human prostate cancer cell lines compared to a BPH cell line (BPH-1 ); see Fig. 3C.
  • ACSS1 acyl-CcA sreE-heta-* -fcart-diam family nyrober 1
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  • A1DH1A5 a ebydfi deii droge iasfi 1 £ ⁇ 4 3 ⁇ 4ily mea-i ei A
  • ANAPC5 ana hase promofirig complex E bsinrt 5
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  • EJF4EBP1 ffiik-rvotic tEsuslatios miti-oasti factor 4E bisdmg protein 1
  • ERGFC1 ⁇ de la-an-c re kr-dij-B-solEi jxsienDeiiiale c®323D3ztw& ⁇ ii (ERGI 1
  • FAM19A4 famil nam. -equence ⁇ molarity 19 (chemokiae (C-C mstiQ-3ike). Bsember A4
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  • LRGUK 1 « ⁇ - ⁇ *» ⁇ > ⁇ SJJIJ roamlate Vma-ae j rain wv»j»im
  • LKIG1 leucine-rieh re eat aj3 ⁇ 4 immnTOgiobnlm-like dems is 1
  • NDUFA3 NADH ckhydrogHiase (iib cjUHKme ⁇ 1 sipiia mtxromplex. 8, l3 ⁇ 4Da 5UFB10 NADH d r drogesa e (ubiquiiKHie ⁇ 1 befe d ocnple . If ⁇ . 22kDs
  • PAOX poi amine oxi fae* ( ⁇ - ⁇ -3 ⁇ )
  • PC A3 prostate cancel' s rigen 3 (najs-proteiE coding)
  • RAB3B RAB3B. member RAS oncogene
  • RG9MTD2 RNA (guanine ⁇ -) rnel-iYltcansferase domain containin 2
  • SIX3SF2 solute carrier family 35. rsens er F2
  • SNO D104 anal nucleolar XNA CD os 104
  • TA52X10 taste- receptor, type 2, membe 10
  • TBC1D3B TBC 1 domain family, member 3B
  • TBC1D4 TBC 1 dosssam family, iisember 4
  • T ED3 tismsioembraiifi emp24 protein transport domain cor-taii-uas 3
  • IMEFF2 transmembrane protein with. EGF-like and two foDi tatsn-like domais: 2
  • VLDLBL very low dsniitj' lipoprotein receptor
  • JES PCAT18-/JUPITER-associated expression signature
  • JUPITER-associated expression signature JES
  • PCAT18 The functional relevance of PCAT18 in PCa cells was then determined. To this aim, PCAT18's expression levels in a panel of prostate cell lines was measured. In keeping with the previous data, PCAT18 expression was higher in AR-positive than in AR- negative PCa cells ( Figure 8(C)). Among AR-positive cells, PCAT18 levels incrementally increased from non-neoplastic (BPH1 ), to androgensensitive (22Rv1 , LNCaP) and androgen- insensitive (C4-2) PCa cells. LNCaP and its castrate-resistant sub-line C4-2 (Wu HC, et al. Derivation of androgen-independent human LNCaP prostatic cancer cell sublines: role of bone stromal cells.
  • EXAMPLE 5 Effect of silencing PCAT18 using siRNAs.
  • siRNAI and siRNA2 Two small-interfering RNAs (siRNAs) (siRNAI and siRNA2, the sequences of which are set out in Table 1 1 ) were used in a human prostate cancer cell line (C4-2) to silence JUPTER/PCAT18 expression. These two siRNAs induced greater than 80% gene knockdown at a 2nM concentration ( Figure 8(E)) (Kim DH, et al. Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nature biotechnology. 2005; 23(2):222-226). PCAT18 silencing (24-48h) significantly inhibited PCa cell invasion and migration ( Figures 7(A) and 7(B); see also Figure 3(D)).
  • siRNAI 22 AGCAGGAACAUUCCAAUAGAAGAAAUAUUGGAAUGU siRNA2 23 GCAACAUGACCUACAGUUAAUGAGUAACUGUAGGUC
  • IncRNAs in the metastatic versus non-metastatic xenografts The most up-regulated transcript was an uncharacterized IncRNA, PCAT18 (also referred to as JUPITER), is characterized herein.
  • PCAT18 is specifically expressed in normal prostate compared to 11 normal tissues (p ⁇ 0.05) and specifically up-regulated in PCa compared to 15 other neoplasms (p ⁇ 0.001). Cancer-specific up-regulation of PCAT18 was confirmed on an independent dataset of PCa and benign prostatic hyperplasia samples (p ⁇ 0.001 ). In addition, PCAT18 was detectable in plasma samples and increased incrementally from normal individuals to those with localized and metastatic PCa (p ⁇ 0.01).
  • PES PCAT18-associated expression signature
  • JES J PCAT18-associated expression signature
  • PCa samples are often composed of multi-clonal subpopulations, each with a different mutational spectrum and metastatic potential (18).
  • Molecular analysis of PCa samples is affected by this heterogeneity, which often masks the aggressive signature of truly metastatic cells.
  • the development of gene expression profile-based diagnostic and prognostic algorithms is particularly challenging in PCa.
  • one tumor tissue line When engrafted in the sub-renal capsule of immunocompromised mice, one tumor tissue line invariably gave rise to localized and poorly-invasive tumors; the other line was reproducibly able to generate highly invasive tumors, producing distant metastases through predictable routes. Since the two tumor tissue lines are derived from the same patient and share most of the genetic alterations with the donor tissue, they represent an ideal model to study gene expression changes related to PCa progression to a metastatic state. A similar model had been successfully exploited for the identification of PCa-associated miRNAs and protein coding genes (10, 14). [00201] Data from 4 independent datasets and more than 600 human samples confirmed .that this gene is prostate-specific and highly up-regulated in PCa.
  • the data herein indicates that PCAT18 is more over-expressed in PCa than PCGEM1 and that a set of patients over-expressing this gene does not express PCA3 (cBio portal, data not shown). Since PCAT18 is so frequently over-expressed in PCa cells and PCa- specific, its measurement in plasma samples (alone or in combination with other non-coding RNAs) can allow earlier and more accurate detection of PCa progression to a metastatic and drug-resistant stage. IncRNA is detectable in plasma samples from PCa patients and can discriminate between localized and mCRPC.
  • RNA maps reveal new RNA classes and a possible function for pervasive transcription. Science.
  • RNA-seq analysis of prostate cancer in the Chinese population identifies recurrent gene fusions, cancer-associated long noncoding RNAs and aberrant alternative splicings. Cell research. 2012;22:806-21.

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Abstract

Selon un aspect, l'invention concerne des procédés et les utilisations de PCAT18 pour diagnostiquer, pronostiquer et surveiller le traitement du cancer de la prostate (PCa) chez un sujet. Selon un autre aspect, l'invention concerne des procédés de traitement du PCa chez un sujet par administration d'un agent d'inhibition de PCAT18. L'invention concerne également les utilisations de PCAT18 pour traiter le PCa chez un sujet, et des compositions pharmaceutiques comprenant un agent thérapeutique efficace pour limiter la quantité de PCAT18 dans les cellules cancéreuses de la prostate et un excipient pharmaceutiquement acceptable. La transcription de PCAT18 est un ARN long non codant (Arnica) dont l'expression est significativement modifiée dans des échantillons biologiques prélevés sur des sujets souffrant du PAC ou risquant de développer un PAC par rapport à des individus normaux. L'expression de PCAT18 est spécifique au tissue prostatique et est élevée à la fois dans un tissu prostatique cancéreux et dans le plasma sanguin de sujets souffrant de PAC par rapport à des sujets sans PAC ou à des patients avec d'autres formes de cancer.
PCT/CA2014/000538 2013-06-28 2014-06-27 Procédés et utilisations pour diagnostiquer et traiter le cancer de la prostate WO2014205555A1 (fr)

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